Phosphitylation process

ABSTRACT

A process for the phosphitylation of an alcohol or thiol with a phosphitylation agent in the presence of an activator is provided. The activator has the formula: 
                         
wherein p is 0 or an integer from 1 to 4 and R for each occurrence is a substituent. Preferably X 7  is O and p is 0. The activator is commonly employed as a salt complex with an organic base. Preferred alcohols or thiols include nucleosides and oligonucleotides. The process is particularly suited for the synthesis of phosphoramidites.

This is a 371 filing based on PCT/GB2003/004312, filed Aug. 10, 2003 andclaims the benefit of U.S. Provisional Application No. 60/418,185, filedOct. 15, 2002.

The present invention concerns a process for the phosphitylation of analcohol or thiol, and particularly the phosphitylation of a nucleosideto form a nucleoside phosphoramidite.

Synthetic oligonucleotides are important diagnostic tools for thedetection of genetic and viral diseases. In addition, oligonucleotidesand modified oligonucleotides are of interest as therapeutic candidatesthat inhibit gene expression or protein function. Large scale synthesisof oligonucleotides for use as therapeutic candidates has becomeincreasingly important since FDA approval of an oligonucleotide analogfor the treatment of cytomegalovirus (CMV), and several otheroligonucleotide analogs are currently in clinical trials. Kilogramquantities of a purified oligonucleotide analog are needed for eachclinical trial.

The principal method currently employed for the preparation ofoligonucleotide is the phosphoramidite approach. The increasing demandfor larger quantities of oligonucleotides has correspondingly increaseddemand for phosphoramidite compounds. Phosphoramidite compounds arecommonly prepared by phosphitylation of a nucleoside with aphosphitylation agent in the presence of an activator. The most commonlyused activator is the nucleophilic activator 1H-tetrazole. However,1H-tetrazole is explosive and therefore can be hazardous to use in largescale syntheses.

Non-explosive activators that promote phosphitylation and which may beemployed without increasing side products are needed in order to makeoligonucleotides more readily available for diagnostic and therapeuticuse.

According to the present invention, there is provided a process for thephosphitylation of an alcohol or thiol with a phosphitylation agent inthe presence of an activator, characterised in that the activator hasthe formula 1:

In formula 1, p is 0 or an integer from 1 to 4. R for each occurrence isa substituent, preferably each independently, a halo, a substituted orunsubstituted aliphatic group, —NR¹R², —OR³, —OC(O)R³, —C(O)OR³, cyano,a substituted or unsubstituted aryl, a substituted or unsubstitutedheterocyclyl, —CHO, —COR³, —NHCOR³, a substituted or unsubstitutedaralkyl, halogenated alkyl (e.g., trifluoromethyl and trichloromethyl),or —SR³.

Preferably, R is halo, a substituted or unsubstituted aliphatic group,—NR¹R², —OR³, —OC(O)R³, —C(O)OR³, or cyano. Alternatively, two adjacentR groups taken together with the carbon atoms to which they are attachedform a six membered saturated or unsaturated ring, preferably anaromatic ring. R¹ and R² are each, independently, —H, a substituted orunsubstituted aliphatic group, a substituted or unsubstituted arylgroup, a substituted or unsubstituted aralkyl group; or together withthe nitrogen to which they are attached form a heterocyclyl group. R³ isa substituted or unsubstituted aliphatic group, a substituted orunsubstituted aryl group, or a substituted or unsubstituted aralkylgroup. X is O or S. Preferably, X is O. It is particularly preferredthat X is O and p is 0.

Preferably the compound of formula 1 is employed as a salt complex withan organic base.

In many embodiments, the alcohol or thiol is a nucleoside oroligonucleotide comprising a free hydroxy or thiol group, and includesnucleosides and oligonucleotides comprising natural nucleoside pentosesugars and unnatural nucleoside sugars, such as hexoses. When thealcohol or thiol is a nucleoside or oligonucleotide, it is often aprotected deoxyribonucleoside, protected ribonucleoside, protectedoligodeoxyribonucleotide, protected oligoribonucleotide or a protectedoligonucleotide having a mixture of deoxyribonucleotide andribonucleotide moieties each comprising a free 3′- or 5′-, preferably a3′-, hydroxy or thiol group, and most preferably a 3′-hydroxy group.

Alcohols and thiols which can be phosphitylated by the process of thepresent invention include compounds having the formula 2:

where A represents

wherein X¹ for each occurrence is, independently, —O— or —S—.Preferably, X¹ is —O— at every occurrence. X² for each occurrence is,independently, —O—, —S—, —CH₂—, or —(CH₂)₂—.

Preferably, X² is —O— at every occurrence. X³ for each occurrence is,independently, O or S. In a more preferred embodiment, X¹ and X² areeach —O— at every occurrence. R⁴ is an alcohol protecting group or athiol protecting group. Preferably, R⁴ is an acid labile protectinggroup. R⁵ for each occurrence is, independently, —H, —F —OR⁶, —NR⁷R⁸,—SR⁹, or a substituted or unsubstituted aliphatic group, such as methylor allyl. R¹⁰ for each occurrence is, independently, a phosphorusprotecting group, commonly a cleavable phosphorus protecting groupemployed in oligonucleotide synthesis, and preferably a substituted orunsubstituted aliphatic group, a substituted or unsubstituted aryl groupor a substituted or unsubstituted aralkyl group, such as a group offormula —CH₂CH₂CN, —CH₂CH₂CN, —CH₂CH₂—Si(CH₃)₂C₆H₅,—CH₂CH₂—S(O)₂—CH₂CH₃, —CH₂CH₂—C₆H₄—NO₂, —CH₂CH₂—Si(CH₃)₂C₆H₅,—CH₂CH₂—S(O)₂—CH₂CH₃, or —CH₂CH₂—C₆H₄—NO₂. R⁶ for each occurrence is,independently, —H, a substituted or unsubstituted aliphatic group (e.g.,methyl, ethyl, methoxyethyl or allyl), a substituted or unsubstitutedaryl group, a substituted or unsubstituted aralkyl, an alcoholprotecting group, especially a base-labile or a silyl protecting group,or —(CH₂)_(q)—NR¹¹R¹². R⁷ and R⁸ for each occurrence are each,independently, —H, a substituted or unsubstituted aliphatic group, or anamine protecting group. Alternatively, R⁷ and R⁸ taken together with thenitrogen to which they are attached are a heterocyclyl group. R⁹ foreach occurrence is, independently, —H, a substituted or unsubstitutedaliphatic group, or a thiol protecting group. R¹¹ and R¹² are each,independently, —H, a substituted or unsubstituted aryl group, asubstituted or unsubstituted heteroaryl group, a substituted orunsubstituted aliphatic group, a substituted or unsubstituted aralkylgroup, a substituted or unsubstituted heteroaralkyl group or an amineprotecting group. Alternatively, R¹¹ and R¹² taken together with thenitrogen to which they are attached form a heterocyclyl group. q is aninteger from 1 to about 6. s is 0 or a positive integer. Preferably, sis 0, 1 or 2, and most preferably 0. Each B, independently, is —H, anatural or unnatural nucleobase, protected nucleobase, protected naturalor unnatural nucleobase, heterocycle or a protected heterocycle.

Nucleoside bases include naturally occurring bases, such as adenine,guanine, cytosine, thymine, and uracil and modified bases such as7-deazaguanine, 7-deaza-8-azaguanine, 5-propynylcytosine,5-propynyluracil, 7-deazaadenine, 7-deaza-8-azaadenine,7-deaza-6-oxopurine, 6-oxopurine, 3-deazaadenosine,2-oxo-5-methylpyrimidine, 2-oxo-4-methylthio-5-methylpyrimidine,2-thiocarbonyl-4-oxo-5-methylpyrimidine, 4-oxo-5-methylpyrimidine,2-amino-purine, 5-fluorouracil, 2,6-diaminopurine, 8-aminopurine,4-triazolo-5-methylthymine, 4-triazolo-5-methyluracil and hypoxanthine.

A protected nucleoside base is a nucleoside base in which reactivefunctional groups of the base are protected. Similarly, a protectedheterocycle is a heterocycle in which reactive substitutents of theheterocycle are protected. Typically, nucleoside bases or heterocycleshave amine groups which can be protected with an amine protecting group,such as an amide or a carbamate. For example, the amine groups ofadenine and cytosine are typically protected with benzoyl protectinggroups, and the amine groups of guanine is typically protected with anisobutyryl group, a 4-isopropylphenoxyacetyl group ort-butylphenoxyacetyl group. However, other protection schemes, such asformamidine, may be used. For example, for fast deprotection, the aminegroups of adenine and guanine are protected with phenoxyacetyl groupsand the amine group of cytosine is protected with an isobutyryl group oran acetyl group. Conditions for removal of the nucleobase or heterocycleprotecting group will depend on the protecting group used. When an amideprotecting group is used, it can be removed by treating theoligonucleotide with a base solution, such as a concentrated ammoniumhydroxide solution, n-methylamine solution or a solution of t-butylaminein ammonium hydroxide.

Amine, hydroxy and thiol protecting groups are known to those skilled inthe art. For examples of amine protecting groups see Greene, et al.,Protective Groups in Organic Synthesis (1991), John Wiley & Sons, Inc.,pages 309-405, the teachings of which are incorporated herein byreference in their entirety. Preferably, amines are protected as amidesor carbamates. For examples of hydroxy protecting groups see Id., pages10-142, the teachings of which are incorporated herein by reference intheir entirety. Examples of protecting groups which may be employedinclude silyl groups, especially trialkyl, for exampletri(C₁₋₄alkyl)silyl groups. A preferred silyl protecting group is at-butyldimethylsilyl group. A preferred hydroxy protecting group ist-butyldimethylsilyl group. For examples of thiol protecting groups seeId., pages 277-308, the teachings of which are incorporated herein byreference in their entirety.

An acid labile protecting group is a protecting group which can beremoved by contacting the group with a Bronsted or a Lewis acid. Acidlabile protecting groups are known to those skilled in the art. Examplesof common acid labile protecting groups include substituted orunsubstituted trityl groups (Id., pages 60-62), substituted orunsubstituted tetrahydropyranyl groups (Id., pages 31-34), substitutedor unsubstituted tetrahydrofuranyl groups (Id., pages 36-37) or pixylgroups (Id., page 65). Trityl groups are commonly substituted byelectron donating substituents such as alkoxy groups. A preferred acidlabile protecting group is a substituted or unsubstituted trityl, forexample 4,4′-dimethoxytrityl (hereinafter “DMT”).

A base labile protecting group is a protecting group which can beremoved by contacting the group with a Bronsted or a Lewis base. Baselabile protecting groups are known to those skilled in the art. Examplesof common base labile protecting groups include carbonyl compounds, suchas acetyl, benzoyl and pivaloyl groups.

It will be recognised that, whilst the formula 2 is expressed in termsof the natural, nucleosidic configuration (D-isomers) of the givenalcohols, the present invention is equally applicable to thecorresponding synthetic or unnatural configuration (L-isomers) of thealcohols, and to mixtures of both configurations.

Phosphitylation agents that can be employed in the process of thepresent invention commonly have the general chemical formula:R¹³—X⁶—PX⁴X⁵wherein R¹³ represents a phosphorus protecting group, commonly acleavable phosphorus protecting group employed in oligonucleotidesynthesis, for example a substituted or unsubstituted aliphatic oraralkyl group, such as a methyl group, —CH₂CH₂—Si(CH₃)₂C₆H₅,—CH₂CH₂—S(O)₂—CH₂CH₃, —CH₂CH₂—C₆H₄—NO₂ and preferably a group of formula—CH₂CH₂CN; a substituted or unsubstituted aromatic group, such as aphenyl or substituted phenyl, for example a 4-chlorophenyl,2-chlorophenyl, 2-nitrophenyl or 4-nitrophenyl group; X⁶ represents O orS, and preferably 0; X⁴ and X⁵, which may be the same of different,represent leaving groups, such as halo, commonly bromo or chloro, or—NR¹⁴R¹⁵, wherein R¹⁴ and R¹⁵ each independently represents an alkyl,preferably a C₁₋₆ alkyl, group, or R¹⁴ and R¹⁵ are joined, together withthe N to which they are attached, to form a 5-7 membered ring. Commonly,at least one of X⁴ and X⁵ is a group of formula —NR¹⁴R¹⁵. Mostpreferably, X⁴ and X⁵ are the same, and it is particularly preferredthat both X⁴ and X⁵ are —N[CH(CH₃)₂]₂ groups. It is especially preferredthat X⁶ is O and R¹³ —CH₂CH₂CN.

The process of the present invention is particularly suited to thepreparation of phosphoramidites, particularly nucleoside oroligonucleotide phosphoramidites.

Examples of preferred phosphitylating agents includeO-β-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite, (commonlyknown as “tetraphos”),O-β-cyanoethyl-N,N,N′,N′-tetramethylphosphorodiamidite,O-β-cyanoethyl-N,N,N′,N′-tetraethylphosphorodiamidite, bis(N,N-diisopropylamino)-2-methyltrifluoroacetylamino-ethoxyphosphine, bis(N,N-diisopropylamino)-2-diphenylmethylsilylethoxyphosphine andO-β-cyanoethyl-bis (N-morpholino) phosphorodiamidite.

The process according to the present invention is often carried out at atemperature in the range of from 0° C. to about 50° C., and preferablyat ambient temperature, such as from about 15° C. to about 30° C.

Advantageously, substantially anhydrous reaction conditions areemployed.

In many embodiments the process of the present invention is carried outunder an inert atmosphere, such as a nitrogen or argon atmosphere.

The process according to the present invention is advantageouslyemployed to produce nucleoside phosphoramidites. Accordingly, apreferred aspect of the present invention comprises a process for thepreparation of a compound of formula:

which comprises reacting a compound of formula:

wherein R⁴ is as previously defined, preferably a dimethoxytrityl group,and R⁵ is as previously defined;with a compound of formula:NCCH₂CH₂O—P(N(R¹⁶)₂)₂wherein R¹⁶ represents a C₁₋₆ alkyl group, preferably an isopropylgroup; in the presence of an activator, where the activator comprises acompound of formula:

and an organic base.

In many embodiments, the activator is employed at a stoichiometric orsub-stoichiometric mole ratio to the alcohol, with mole ratios ofactivator to alcohol of from about 0.4:1 to 1:1, particularly from about0.5:1 to 0.75:1 being preferred.

The phosphitylating agent is often employed at a stoichiometric moleratio to the alcohol, or in excess, with mole ratios of phosphitylatingagent to alcohol of from about 1:1 to 3:1, particularly from about 1:1to 1.5:1, being preferred.

In the presence of an organic base, the activators employed in thepresent invention have good solubility particularly in organic solventsthat are typically used for phosphitylation. The concentration of theactivator and the organic base can be up to the solubility of theactivator in the solvent concerned. In a preferred embodiment, theactivator and the organic base are present in a concentration range ofabout 0.01 M to about 2M, for example from about 0.05M to about 0.5M.Commonly, the activator and the organic base are present at aconcentration of up to 0.25M, such as from about 0.1M to about 0.25M. Ina more preferred embodiment, the activator and the organic base arepresent in the same molar concentration. In certain embodiments, theorganic solvent is a chlorocarbon, such as dichloromethane. In apreferred embodiment, the organic solvent comprises acetonitrile. Inanother preferred embodiment, the organic solvent comprises an organicamide, such as dimethylformamide, 1-methyl-2-pyrrolidinone or1,3-dimethyl-2-imidazolidinone.

An organic base is an organic compound that has a tendency to acceptprotons at pH 7. Preferred organic bases are secondary amines, tertiaryamines or azaheterocyclyl compounds, each of which may be substituted orunsubstituted by one or more substituents. An aprotic organic base is anorganic base that has no hydrogen bonding protons in its chemicalstructure before accepting a proton. Aprotic organic bases such astertiary amines and aprotic azaheterocyclyl compounds are preferablyused in conjunction with compounds of formula 1, as described herein, topromote phosphitylation reactions.

Azaheterocyclyl compounds, as used herein, include heteroaryl groupswhich have one or more nitrogen atom in the aromatic ring andheteroalicyclyl groups that have at least one nitrogen atom in thenon-aromatic ring system. Preferably, azaheteroaryl compounds have five-or six-membered aromatic rings with from one to three nitrogens in thearomatic ring. Preferably, azaheteroalicyclyl compounds are five- orsix-membered rings, commonly comprising one or two nitrogens in thering. Preferred azaheterocyclyl compounds are organic bases. Examples ofazaheterocyclyl compounds that are organic bases include pyrimidines,1-alkylpyrazoles, especially 1-(C₁₋₄ alkyl)pyrazoles, 1-arylpyrazoles,1-benzylpyrazoles, pyrazines, N-alkylpurines, especially N-(C₁₋₄alkyl)purines, N-arylpurines, N-benzylpurines, N-alkylpyrroles,especially N-(C₁₋₄ alkyl)pyrroles, N-arylpyrroles, N-benzylpyrroles,pyridines, N-alkylimidazoles, especially N-(C₁₋₄ alkyl)imidazoles,N-arylimidazoles, especially N-phenylimidazole, N-benzylimidazoles,quinolines, isoquinolines, quinoxalines, quinazolines, N-alkylindoles,especially N-(C₁₋₄ alkyl)indoles, N-arylindoles, N-benzylindoles,N-alkylbenzimidazoles especially N-(C₁₋₄ alkyl)benzimidazoles,N-arylbenzimidazoles, N-benzylbenzimidazoles, triazine, thiazole,1-alkyl-7-azaindoles, especially 1-(C₁₋₄ alkyl)-7-azaindoles,1-aryl-7-azaindole 1-benzyl-7-azaindoles, pyrrolidines, morpholines,piperidines, and piperazines. Especially preferred azaheterocyclylcompounds are pyridines, such as pyridine and 3-methylpyridine, andN-(C₁₋₄ alkyl) imidazoles, such as N-methylimidazole.

Tertiary amines are organic bases that have a nitrogen atom which isbonded to three carbon atoms, often to three aryl, commonly phenyl,and/or alkyl groups, commonly to three alkyl groups, including forexample trialkylamines such as trimethylamine, triethylamine, anddiisopropylethylamine. In addition, tertiary amines can beazaheterocyclyl groups wherein the nitrogen atom is aprotic. Tertiaryamines that are azaheterocyclyl groups are preferred. Examples ofazaheterocyclyl tertiary amines are N-alkylpyrrolidines,N-arylpyrrolidines, N-alkylpyrroles, N-arylpyrroles, N-alkylmorpholines,N-arylmorpholines, N-alkylpiperidines, N-arylpiperidines,N,N-dialkylpiperazines, N,N-diarylpiperazines,N-alkyl-N-aryl-piperazines, quinuclidines,1,5-diazabicyclo[4.3.0]non-5-enes and1,8-diazabicyclo[5.4.0]undec-7-enes. Tertiary amines can also beazaheteroaryl or azaheteroalicyclyl compounds.

Secondary amines are organic bases comprising a nitrogen bonded to asingle hydrogen and to two carbon atoms. Commonly the nitrogen atom isbonded to two alkyl or aryl groups or forms part of an azaheterocyclicgroup. Examples of secondary amine compounds include diethylamine anddiisopropylamine.

Particularly preferred organic bases include pyridine, 3-methylpyridine,and N-methylimidazole.

Suitable substituents for aliphatic groups, aryl groups, aralkyl groups,heteroaryl groups, azaheteroaryl groups and heteroalicyclyl groupsinclude aryl groups, halogenated aryl groups, alkyl groups, halogenatedalkyl (e.g. trifluoromethyl and trichloromethyl), aliphatic ethers,aromatic ethers, benzyl, substituted benzyl, halogens, particularlychloro and fluoro groups, cyano, nitro, —S-(aliphatic or substitutedaliphatic group), and —S-(aromatic or substituted aromatic).

Aliphatic groups, as used herein, include straight chained or branchedC₁-C₁₋₁₈ hydrocarbons which are completely saturated or which containone or more unconjugated double bonds, or cyclic C₃-C₁₈ hydrocarbonswhich are completely saturated or which contain one or more unconjugateddouble bonds. Alkyl groups are straight chained or branched C₁-C₈hydrocarbons or C₃-C₈ cyclic hydrocarbons which are completelysaturated. Aliphatic groups are preferably alkyl groups.

Aryl groups include carbocyclic aromatic ring systems (e.g., phenyl) andcarbocyclic aromatic ring systems fused to one or more carbocyclicaromatic (e.g., naphthyl and anthracenyl) or an aromatic ring systemfused to one or more non-aromatic ring (e.g.,1,2,3,4-tetrahydronaphthyl).

Heterocyclic groups, as used herein, include heteroaryl groups andheteroalicyclyl groups. Heteroaryl groups, as used herein, includearomatic ring systems that have one or more heteroatoms selected fromsulfur, nitrogen or oxygen in the aromatic ring. Preferably, heteroarylgroups are five or six membered ring systems having from one to fourheteroatoms. A heteroalicyclyl group, as used herein, is a non-aromaticring system that preferably has five to six atoms and includes at leastone heteroatom selected from nitrogen, oxygen, and sulfur. Examples ofheterocyclic groups include morpholinyl, piperidinyl, piperazinyl,thiomorpholinyl, pyrrolidinyl, thiazolidinyl, tetrahydrothienyl,azetidinyl, tetrahydrofuryl, dioxanyl and dioxepanyl thienyl, pyridyl,thiadiazolyl, oxadiazolyl, indazolyl, furans, pyrroles, imidazoles,pyrazoles, triazoles, pyrimidines, pyrazines, thiazoles, isoxazoles,isothiazoles, tetrazoles, oxadiazoles, benzo(b)thienyl, benzimidazole,indole, tetrahydroindole, azaindole, indazole, quinoline,imidazopyridine, purine, pyrrolo[2,3-d]pyrimidine, andpyrazolo[3,4-d]pyrimidine.

An aralkyl group, as used herein, is an aromatic substituent that islinked to a moiety by an alkyl group. Preferred aralkyl groups includebenzyl groups.

A heteroaralkyl group, as used herein, is a heteroaryl substituent thatis linked to a moiety by an alkyl group.

The present invention is illustrated without limitation by the followingExamples.

EXAMPLE 1

The N-methylimidazole salt of saccharin was prepared by the followingprocedure. Saccharin was suspended in acetonitrile, and 1.1 eq. ofN-methylimidazole with respect to the saccharin was added dropwise tothe suspension. The reaction mixture was concentrated under reducedpressure to form the crystalline salt which was washed with either etheror hexane to remove traces of N-methylimidazole and acetonitrile.

EXAMPLE 2

A series of nucleosides was phosphitylated usingO-β-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoramidite and theN-methylimidazole salt of saccharin as activator.

General Method:

In an appropriate sized flask was added the nucleoside (1.5 mmol) andthe solid was dried azeotropically by distilling (rotary evaporator) twotimes with 20 mL of pyridine. The flask was purged with Ar and to theflask was added 15 mL of acetonitrile. The mixture was stirred at roomtemperature until a clear solution was obtained. To the mixture wasadded O-β-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoramidite (Tetraphos)followed by the addition of N-methylimidazole salt of saccharin. Themixture was stirred at room temperature while the reaction was monitoredfor end of reaction by HPLC. At the end of the reaction, the mixture wasdiluted with 30 mL of ethyl acetate and the organic mixture was washedwith 2×25 mL of saturated aqueous sodium bicarbonate and 25 mL ofsaturated aqueous sodium chloride. The organic layer was separated anddried over MgSO₄. The suspension was filtered and the solvent wasremoved using a rotary evaporator. The residue was dried under vacuum togive a foam.

TABLE 1 Results of amidite synthesis Rxn Tetraphos Activator time %amidite^(a) yield^(b) (eq.) (eq.) nucleoside (h) (HPLC) (%) 1.2 0.65′-DMT-N-Bz-deoxyA 7 91.9 84 1.2 0.5 5′-DMT-N-Bz-deoxyA 5 91.5 86 1.21.0 5′-DMT-N-Bz-deoxyA 5 89.8 87 1.2 0.5 5′-DMT-N-iBu-deoxyG 16 79.1 851.2 0.5 5′-DMT-N-Ac-2′-OMeC 5 89.3 85 1.2 0.6 5′-DMT-N-Bz-deoxyC 8 91.179 1.2 0.6 5′-DMT-2′-TBDMS-L-U 16 82.6 82 1.2 0.65′-DMT-N-iBu-2′-TBDMS-L-G 16 59.3 84 2.2 0.6 5′-DMT-N-iBu-2′-TBDMS-L-G16 87.4 82 ^(a)% amidite = % amidite in crude product ^(b)yield = yieldof crude product

EXAMPLE 2

In a 500 mL round bottom flask was added 5′-DMT-N-Bz-2′-deoxyadenosine(18.00 g, 27.37 mmol) and the solid was dried azeotropically by theaddition and evaporation (rotary evaporator) of 2×200 mL of toluene. Theresidue was dried under vacuum for 16 h. The residue was dissolved inacetonitrile (180 mL) under an argon atmosphere andO-β-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (9.90 g, 32.84mmol) was added. The mixture was stirred for 5 minutes and solidN-methylimidazole salt of saccharin (3.63 g, 13.69 mmol) was added. Themixture was stirred at room temperature while the reaction was monitoredby HPLC. After 18 h, no further reaction was observed. To the reactionmixture was added ethyl acetate (200 mL)and the organic solution waswashed with saturated aqueous sodium bicarbonate (2×150 mL) andsaturated aqueous sodium chloride (150 mL). The organic layer wasseparated and dried over MgSO₄. The suspension was filtered and thesolvent was removed using a rotary evaporator. The residue was driedunder vacuum for 16 h to give a white foam.

Crude yield: 23.70 g

HPLC: 92.5%

The crude material (23.70 g) was chromatographed using a silica gel (230g) column. The column was loaded using 30% ethyl acetate/hexanescontaining 0.5% triethylamine. The column was washed with 2 columnvolumes of 30% ethyl acetate/hexanes. The crude material was loaded andthe column was eluted using 2 column volumes of 30% ethylacetate/hexanes, 2 column volumes of 40% ethyl acetate/hexanes, 2 columnvolumes of 50% ethyl acetate/hexanes and finally 3 column volumes of 70%ethyl acetate/hexanes. Fractions were collected when the product wasdetected by TLC (8:3 ethyl acetate:hexanes). Fractions containing thedesired product were combined and the solvent was removed using a rotaryevaporator. The residue was dried under vacuum for 16 h to give a whitefoam.

Yield: 17.55 g (75%)

HPLC: 97.5%

³¹P NMR: 99.3%

1. A process for the phosphitylation of an alcohol or thiol with aphosphitylation agent in the presence of an activator, characterised inthat the activator has the formula 1:

wherein p is 0 or an integer from 1 to 4, R for each occurrence is asubstituent, and X⁷ is O or S.
 2. A process according to claim 1,wherein X⁷ is O and p is
 0. 3. A process according to claim 1 or 2,wherein the compound of formula 1 is employed as a salt complex with anorganic base.
 4. A process according to claim 3, wherein the organicbase is selected from the group consisting of pyridine,3-methylpyridine, and N-methylimidazole.
 5. A process according to claim3, wherein the alcohol or thiol is a nucleoside or oligonucleotidecomprising a free hydroxy or thiol group.
 6. A process according toclaim 5, wherein a nucleoside comprising a free 3′-hydroxy group isphosphitylated.
 7. A process according to claim 3, wherein thephosphitylation agent has the general chemical formula:R¹³—X⁶—PX⁴X⁵ wherein R¹³ represents a phosphorus protecting group, X⁶represents O or S, X⁴ and X⁵, which may be the same of different,represent leaving groups.
 8. A process according to claim 7, wherein R¹³represents a substituted or unsubstituted aliphatic or aralkyl group ora substituted or unsubstituted aromatic group, X⁶ is O and X⁴ and X⁵each independently represent —NR¹⁴R¹⁵, wherein R¹⁴ and R¹⁵ eachindependently represents a C₁₋₆ alkyl, group, or R¹⁴ and R¹⁵ are joined,together with the N to which they are attached, to form a 5-7 memberedring.
 9. A process according to claim 8, wherein the phosphitylatingagent is selected from the group consisting ofO-β-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite,O-β-cyanoethyl-N,N,N′,N′-tetramethylphosphorodiamidite,O-β-cyanoethyl-N,N,N′,N′-tetraethylphosphorodiamidite, bis(N,N-diisopropylamino)-2-methyltrifluoroacetylamino-ethoxyphosphine, bis(N,N-diisopropylamino)-2-diphenylmethylsilylethoxyphosphine andO-β-cyanoethyl-bis (N-morpholino) phosphorodiamidite.
 10. A process forthe preparation of a compound of formula:

which comprises reacting a compound of formula:

with a compound of formula:NCCH₂CH₂O—P(N(R¹⁶)₂)₂ in the presence of an activator, where theactivator comprises a compound of formula:

and an organic base, wherein R⁴ is an alcohol protecting group, R⁵ is—H, —F —OR⁶, —NR⁷R⁸, —SR⁹, or a substituted or unsubstituted aliphaticgroup, such as methyl or allyl, R⁶ for each occurrence is —H, asubstituted or unsubstituted aliphatic group, a substituted orunsubstituted aryl group, a substituted or unsubstituted aralkyl, analcohol protecting group, or —(CH₂)_(q)—NR¹¹R¹², R⁷ and R⁸ are each,independently, —H, a substituted or unsubstituted aliphatic group, or anamine protecting group or R⁷ and R⁸ taken together with the nitrogen towhich they are attached are a heterocyclyl group, R⁹ is —H, asubstituted or unsubstituted aliphatic group, or a thiol protectinggroup, R¹¹ and R¹² are each, independently, —H, a substituted orunsubstituted aryl group, a substituted or unsubstituted heteroarylgroup, a substituted or unsubstituted aliphatic group, a substituted orunsubstituted aralkyl group, a substituted or unsubstitutedheteroaralkyl group or an amine protecting group or R¹¹ and R¹² takentogether with the nitrogen to which they are attached form aheterocyclyl group, q is an integer from 1 to about 6, B is —H, anatural or unnatural nucleobase, protected nucleobase, protected naturalor unnatural nucleobase, heterocycle or a protected heterocycle and R¹⁶represents a C₁₋₆ alkyl group, preferably an isopropyl group.
 11. Aprocess according to claim 10, wherein the organic base is selected fromthe group consisting of pyridine, 3-methylpyridine, andN-methylimidazole.
 12. A process according to claim 10 or 11, wherein R⁵is H, OMe or OCH₂CH₂OMe.
 13. A process according to claim 10 or 11,wherein R⁴ is an acid-labile protecting group and R⁵ is OR⁶ wherein R⁶is a base labile protecting group or a silyl protecting group.